Feline herpesvirus 1 (FHV-1) enters the cell by receptor-mediated endocytosis

Abstract

Feline herpesvirus type 1 (FHV-1) is an enveloped dsDNA virus belonging to the Herpesviridae family and is considered one of the two primary viral etiological factors of feline upper respiratory tract disease. In this study, we investigated the entry of FHV-1 into host cells using two models: the AK-D cell line and primary feline skin fibroblasts (FSFs). We employed confocal microscopy, siRNA silencing, and selective inhibitors of various entry pathways. Our observations revealed that the virus enters cells via pH and dynamin-dependent endocytosis, as the infection was significantly inhibited by NH₄Cl, bafilomycin A1, dynasore, and mitmab. Additionally, genistein, nystatin, and filipin treatments, siRNA knock-down of caveolin-1, as well as FHV-1 and caveolin-1 colocalization suggest the involvement of caveolin-mediated endocytosis during the entry process. siRNA knock-down of clathrin heavy chain and analysis of virus particle colocalization with clathrin indicated that clathrin-mediated endocytosis also takes part in the primary cells. This is the first study to systematically examine FHV-1 entry into host cells, and for the first time, we describe FHV-1 replication in AK-D and FSFs. IMPORTANCE Feline herpesvirus 1 (FHV-1) is one of the most prevalent viruses in cats, causing feline viral rhinotracheitis, which is responsible for over half of viral upper respiratory diseases in cats and can lead to ocular lesions resulting in loss of sight. Although the available vaccine reduces the severity of the disease, it does not prevent infection or limit virus shedding. Despite the clinical relevance, the entry mechanisms of FHV-1 have not been thoroughly studied. Considering the limitations of commonly used models based on immortalized cells, we sought to verify our findings using primary feline skin fibroblasts, the natural target for infection in cats.

Keywords: FHV; feline herpesvirus 1; herpesviruses; viral entry; virus-host interactions.

Fig 1

FHV-1 replicates in feline cells. (A) Infections with FHV-1 at MOI = 0.001 and MOI = 1 were carried out using primary FSF, feline fetal lung cells (AK-D), and CRFK cells. Samples were collected at different time points, subjected to viral DNA isolation followed by qPCR, and are presented as a mean of the number of FHV-1 DNA copies/mL ± SD from at least three independent biological repeats. Data were analyzed with Shapiro-Wilk and Brown-Forsythe tests. The Kruskal-Wallis test with post hoc Dunn’s test was used to determine the significance of differences between compared means. Values statistically significant are indicated by asterisks: *P < 0.05, ***P < 0.001, ****P < 0.0001. (B) FHV-1 replication was also confirmed using confocal microscopy. Blue color denotes nuclei, red F-actin, and green FHV-1 virions. Representative images are shown as maximum projections of XY stacks. Scale bar: 10 µm.

Fig 2

FHV-1 glycoproteins expression and interaction with cell host surface. (A) The scheme of expression constructs. (B) Expression of viral glycoproteins and western blot analysis. Truncated FHV-1 glycoprotein B (gB), glycoprotein C (gC), and glycoprotein D (gD) secreted from insect cells were analyzed using anti-HA-Tag antibodies. (C) The adhesion of gC and gD to the cell surface was analyzed by confocal microscopy. Immunostaining was performed as described in the Materials and Methods section. (D) Adhesion of gC to AK-D cells in the presence or absence of soluble heparan sulfate (HS; 1 mg/mL, 1 h at 4°C), as visualized using confocal microscopy. (E) FHV-1 entry assay in the presence or absence of soluble HS (1 mg/mL), as analyzed by qPCR. The blue color denotes nuclei, red F-actin, and green glycoproteins. Representative images are shown as maximum projections of XY stacks and XZ stacks. Scale bar: 10 µm. Data are presented as mean ± SD from three independent biological repeats with three technical replicates each. Data were analyzed with Shapiro-Wilk and Brown-Forsythe tests. One-way ANOVA with post hoc Dunnett’s test was used to determine the significance of differences between compared means. Values statistically significant are indicated by asterisks: ****P < 0.0001.

Fig 3

FHV-1 entry is dynamin-dependent. (A) First, the cytotoxicity of compounds was determined by the XTT assay. (B) For the entry assay, cells were incubated with dynasore or mitmab. Samples were collected at 8 hpi, then subjected to viral DNA isolation followed by qPCR analysis. The data were normalized to the untreated virus-infected cells. (C) Additionally, after an entry assay, infected cells were fixed and immunostained. The blue color denotes nuclei and the green is FHV-1. Representative images are shown. Scale bar: 200 µm. Data are presented as a mean ± SD from three independent experiments, each performed in triplicate or quadruplicate. Data were analyzed with Shapiro-Wilk and Brown-Forsythe tests. One-way ANOVA with post hoc Dunnett’s test was used to determine the significance of differences between compared means. Values statistically significant are indicated by asterisks: ****P < 0.0001.

Fig 4

FHV-1 enters the cell by pH-dependent endocytosis. (A) Virus-treated cells were synchronized at 4°C for 30 min and then incubated at 37°C before being washed and fixed. Immunostaining was performed as described in the Materials and Methods section. Images present the colocalization of EEA1 and the virus after the 30-min incubation at 37°C with either FSFs or AK-D. The blue color denotes nuclei, red FHV-1, and green EEA1. Representative images are shown as maximum projections of XY stacks. Scale bar: 10 µm. Colocalization between FHV-1 and EEA1 was additionally marked by the arrows. (B) The confocal images were also quantified to estimate the colocalization determined by the Pearson’s and Mander’s coefficients for AK-D (green line) and FSFs (red line). (C) The cytotoxicity of compounds was determined by the XTT assay. (D) For the entry assay, cells were incubated with NH₄Cl or bafilomycin A1. Samples were collected at 8 hpi, then subjected to viral DNA isolation followed by qPCR analysis. The data were normalized to the untreated virus-infected cells. Data are presented as a mean ± SD from three independent experiments performed in triplicate or quadruplicate. Data were analyzed with Shapiro-Wilk and Brown-Forsythe tests. One-way ANOVA with post hoc Dunnett’s test was used to determine the significance of differences between compared means. Values statistically significant are indicated by asterisks: **P < 0.01, ****P < 0.0001.

Fig 5

FHV-1 entry is caveolin-1 dependent. (A) Cells were cooled down and infected with FHV-1, the entry was synchronized at 4°C for 30 min, then cells were incubated at 37°C before being washed and fixed. Images represent the colocalization of caveolin-1 and the virus after the 30-min incubation at 37°C with either FSFs or AK-D. The blue color denotes nuclei, red FHV-1, and green caveolin-1. Representative images are shown as maximum projections of XY stacks. Scale bar: 10 µm. Colocalization between FHV-1 and caveolin was additionally marked by the arrows. (B) Quantification of the colocalization was determined by the Pearson’s and Mander’s coefficients for both AK-D (green line) and FSFs (red line), calculated for at least 10 different stacks. (C) Determination of caveolin-1 silencing efficiency using western blotting. The FHV-1 infection in AK-D (D) or FSFs (E) with silenced caveolin 1 expression was determined using qPCR. The data were normalized to the untreated virus-infected cells. Data are presented as a mean ± SD from three independent experiments, each performed in triplicate or quadruplicate. Data were analyzed with Shapiro-Wilk and Brown-Forsythe tests. One-way ANOVA with post hoc Dunnett’s test was used to determine the significance of differences between compared means. Values statistically significant are indicated by asterisks: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 6

FHV-1 entry and replication are hampered by caveolin-1 inhibitors. (A) First, the cytotoxicity of compounds was determined by the XTT assay. (B) For the entry assay, cells were incubated with genistein, nystatin, or filipin III. Samples were collected at 8 hpi, then subjected to viral DNA isolation followed by qPCR analysis. The data were normalized to the untreated virus-infected cells. (C) Additionally, after an entry assay, infected cells were fixed and immunostained. The blue color denotes nuclei and the green is FHV-1. Representative images are shown, and the same images are being used as representatives of the experimental controls as in Fig. 3C. Scale bar: 200 µm. Data are presented as a mean ± SD from three independent experiments, each performed in triplicate or quadruplicate. Data were analyzed with Shapiro-Wilk and Brown-Forsythe tests. One-way ANOVA with post hoc Dunnett’s test was used to determine the significance of differences between compared means. Values statistically significant are indicated by asterisks: ****P < 0.0001.

Fig 7

FHV-1 entry to FSFs is clathrin-dependent but not to AK-D cells. (A) Cells were cooled down and infected with FHV-1, the entry was synchronized at 4°C for 30 min, then cells were incubated at 37°C before being washed and fixed. Images represent the colocalization of clathrin and the virus after the 30-min incubation at 37°C with either FSFs or AK-D. The blue color denotes nuclei, red FHV-1, and green clathrin. Representative images are shown as maximum projections of XY stacks. Scale bar: 10 µm. Colocalization between FHV-1 and clathrin was additionally marked by the arrows. (B) Quantification of the colocalization was determined by the Pearson’s and Mander’s coefficients for both AK-D (green line) and FSFs (red line), calculated for at least 10 different stacks. (C) Determination of clathrin silencing efficiency using western blotting. The FHV-1 infection in AK-D (D) or FSFs (E) with silenced clathrin expression was determined using qPCR. The data were normalized to the untreated virus-infected cells. Data are presented as a mean ± SD from three independent experiments, each performed in triplicate or quadruplicate. Data were analyzed with Shapiro-Wilk and Brown-Forsythe tests. One-way ANOVA with post hoc Dunnett’s test was used to determine the significance of differences between compared means. Values statistically significant are indicated by asterisks: **P < 0.01, ***P < 0.001.